Polyethylene Glycol Precipitation Step Research Articles

8 Suspected presence of macroprolactin in the plasma of horses and ponies

Prolactin is a peptide hormone that plays a role in various physiological functions and its production is influenced by several factors (thyrotropin-releasing hormone, dopamine, season, meal feeding or physiological state such as lactation). Plasma levels help understand reproductive physiology; but may also be a useful indicator of dopamine production at a central level (connection between the hypothalamus and pituitary gland). In human medicine, when prolactin is measured using sandwich immunometric methodologies in automated analysers, hyperprolactinemia diagnosis may be confounded by the presence of a high molecular mass prolactin/IgG autoantibody complex termed macroprolactin, which does not have biological effects. Macroprolactinaemia has not previously been described in horses. Previously equine prolactin has been measured by radioimmunoassay (RIA) analysis; however, more rapid ELISA assays are now available without the radioactive tracer health and safety considerations. In this study, prolactin was measured in plasma from 20 normal horses and 20 horses with pituitary pars intermedia dysfunction, using a commercial ELISA assay (Biorbyt, UK). Surprisingly, while most showed values below 100 ng/mL, 13 appeared to have extremely high concentrations, well in excess of the upper limit of the standard curve at 1000 ng/mL. There was no significant association of this finding with disease category, breed, sex or age. To determine whether the presence of macroprolactin could be causing these aberrant values, a polyethyleneglycol (PEG) precipitation step was introduced, as previously shown in humans, to remove the macroprolactin while leaving the monomeric form in solution. After the addition of PEG and centrifugation to precipitate out macroprolactin, the supernatant with the unprecipitated prolactin was tested and compared with unprecipitated samples from the same individuals. PEG precipitation resulted in a reduction in prolactin values by more than 99% in those with apparent hyperprolactinaemia (to below 200 ng/mL in all cases except one at 243 ng/mL). In the other samples the values were reduced from 67.4 ± 9.9ng/ml to 10.6 ± 0.7ng/ml (approximately 75% reduction). Although this data is preliminary and more validation work is necessary, these findings are consistent with macroprolactin being detected by the ELISA assay in these individual horses. While it may be possible to validate the ELISA assay for measuring prolactin in equine plasma, a PEG precipitation step is required to remove this interfering substance. Further research is needed to confirm the presence and size characteristics of this macroprolactin complex in equine plasma.

Isolation of Nile crocodile (Crocodylus niloticus) serum immunoglobulin M and Y (IgM and IgY)

Crocodile immunity has not been fully characterised with more studies on crocodile innate immunity than cell-mediated or humoral immunity. Crocodile immunoglobulin genes have been described but immunoglobulin proteins have not been isolated or studied biochemically. Two large proteins proposed to be crocodile IgM and IgY were isolated and purified from Crocodylus niloticus sera using two different protocols. A 50% (w/v) ammonium sulfate and a 15% (w/v) polyethylene glycol precipitation step was followed by Cibacron blue F3GA affinity- and Sephacryl-S300 gel filtration chromatography. An alternate purification protocol, with only two steps, involved thiophilic affinity- and Sephacryl-S300 gel filtration chromatography. The purified crocodile IgM resolved on reducing SDS-PAGE with an apparent mass of 180 kDa. Purified crocodile IgY resolved at 180 kDa alongside chicken IgY on a non-reducing SDS-PAGE gel, and is deduced to consist of two 66 kDa heavy and two 23 kDa light chains under reducing conditions. The thiophilic/gel filtration two-step protocol gave three-fold higher yields of isolated protein than the four-step precipitation/chromatography protocol. Antibodies against the isolated crocodile IgM and IgY were raised in chickens and affinity purified. The chicken antibodies differentiated between crocodile IgM and IgY and have the potential for use in the diagnosis of crocodile infections. The purified crocodile antibodies can be biochemically characterised and compared to mammalian and avian antibodies to give a better understanding of crocodile humoral immunity.

The use of polymerase chain reaction in plasma pools for the concomitant detection of hepatitis C virus and HIV type 1 RNA.

Detection of viral nucleic acids might increase blood transfusion safety through the detection of recently infected blood donors during the preseroconversion window period. Individual screening is difficult to apply, because of technical and financial constraints. A polymerase chain reaction (PCR)-based assay including a polyethylene glycol precipitation step was developed for the concomitant detection of hepatitis C virus (HCV) and HIV type 1 (HIV-1) RNA in plasma pools corresponding to 50 blood donations by the use of commercial assays. The assay had a sensitivity of less than 33 copies per mL for HCV RNA and 1000 copies per mL for HIV-1 RNA for each individual sample included in the pool. The eight preseroconversion samples with HCV RNA between 1,250 and 762,000 copies per mL were all detected when 100-microL aliquots from the samples were introduced into 5-mL pools of 50 blood donations. A PCR-based pooling assay associating a prepurification step with polyethylene glycol allows for the screening of blood donations for HCV and HIV-1 RNA without marked loss of sensitivity from that seen with commercially available assays. This procedure might increase blood safety through systematic screening of blood donations at relatively low cost.

A radioimmunoprecipitation polyethylene glycol assay for circulating Entamoeba histolytica antigens

An assay capable of detecting circulating Entamoeba histolytica antigens in amoebiasis is described. This assay utilised a radiolabelled affinity purified rabbit anti- E. histolytica antibody that had been depleted of antibodies that cross-react with human serum proteins, and a polyethylene glycol precipitation step.

A simple and rapid radioimmunoassay for plasma bradykinin

A radioimmunoassay for plasma bradykinin was developed, in which a polyethyl-eneglycol precipitation step was used for the separation of bradykinin from cross-reacting proteins. This method offers the advantage of a high recovery of bradykinin after precipitation of plasma proteins, reproducibility and ease of performance of many samples simultaneously.

37] Detection and quantitation of immune complexes with a rapid polyethylene glycol precipitation complement consumption method (PEG-CC)

Publisher Summary This chapter discusses the detection and quantitation of immune complexes (IC) with a rapid polyethylene glycol precipitation complement consumption method (PEG-CC). Some assays make use of the isolation of IC through a polyethylene glycol precipitation step. This approach for physicochemical isolation of IC is combined with a simple one-tube, two-step complement consumption procedure for the quantitation of the precipitated and redissolved IC. The precipitable IC activity amounted to 90%-95% after 60 min and 95%-100% after 90 min, as compared to the values obtained after incubation at 4° for 18 hr. For practical reasons, 90 min was chosen for the standardized assay. There were no convincing differences in the reactivity of normal and patient sera or in the precision, when the precipitate was washed with 1ml of 3.0–5.0% PEG and redissolved for 30 min at 22°. The PEG-CC assay has been used to monitor a variety of patient categories and high concentrations of IC have been found in acute SLE, primary biliary cirrhosis, mixed cryoglobulinemia, and in more than 90% of the patients with active classical sero-positive rheumatoid arthritis.

A simple method for the preparation of large quantities of pure plasmid DNA

Polyethylene glycol quantitatively precipitates plasmid DNA of molecular weight 6–123 · 10 6, from cleared lysates of plasmid-carrying bacterial strains. After resuspension and density-gradient centrifugation of the precipitated DNA, it is unchanged in length and in transformation efficiency for Escherichia coli K12. Plasmid DNA can be easily prepared in large quantities by including a polyethylene glycol precipitation step in standard plasmid isolation procedures.

Identification of two distinct heparin cofactors in human plasma: Separation and partial purification

Two distinct components having potent heparin cofactor activity have been separated from human blood plasma and partially purified by means of precipitation with polyethylene glycol, adsorption on Al(OH) 3 gel and ion exchange chromatography on DEAE-cellulose and hydroxyapatite. The first component, cofactor A, is distinguished from the second, cofactor B, by its very low activity in the progressive antithrombin assay. Ion exchange rechromatography experiments indicate that cofactors A and B are not interconvertible, and that neither is an artifact of the polyethylene glycol precipitation step. Molecular weights, estimated by gel filtration were found to be 105,000 and 87,000 daltons for cofactors A and B respectively. Except for the molecular weight, the bulk of evidence suggests that cofactor B and the antithrombin III purified by Abildgaard (3) are identical.